MODULATION OF PROTEIN N-MYRISTOYLATION IN MYCO PATHOGENS

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Covalent attachment of the 14 carbon saturated fatty acid - myristic acid - to the NH2-terminal Gly residue of a number of cellular and viral proteins is critical to their function. Our group has been using biochemical, organic and chemical and molecular genetic approaches to understand the substrate specificity and mechanism of action of the enzyme which catalyzes this modification - myristoyl CoA:Protein N-myristoyltranserase (NMT, E.C. 2.3.1.97) - as well as the physical chemical properties of myristic acid which are important for fulfilling its biological function in N- myristoylated proteins. During the course of these studies we have synthesized heteroatom containing analogs of myristate with altered hydrophobicity which (i) are alternative substrates for mammalian (human) NMTs; (ii) are selectively incorporated into subsets of cellular and viral N-myristoyl proteins (including the HIV-I Pr55gag); and (iii) inhibit HIV replication in an acute H9 T cell assay without accompanying cellular toxicity. We have also cloned the MNT gene from S. cerevisiae and shown that removing NMT from the cell (through gene disruption or deletion) is lethal to the organism. Finally, differences in the substrate specificities of yeast and mammalian NMTs have been identified. The overall purpose of this grant proposal is to develop compounds which will exhibit species specific inhibition of fungal NMTs. The goals are to isolate NMT from C. albicans - a common human pathogen in HIV-I infected individuals. The acyl CoA and peptide substrate specificities of C. albicans and human NMTs will be compared. We will isolate the C. albicans gene and express it in E. coli using a host vector system already used for expression of S. cerevisiae NMT. This will provide a continuing source of large amounts of material for enzymatic as well as structural studies. A rapid, well characterized in vitro assay system will be employed to screen several different classes of compounds as specific inhibitors of C. albicans NMT. These include (i) a large panel of analogs of myristic acid with systematically varied structural features; (ii) transition state compounds; (iii) synthetic peptides and (iv) natural products. The proposed project involves a multidisciplinary team, composed of members of two universities and the Monsanto Company that has worked with one another over the past 3 years on protein N-myristoylation.